企业QQ
官方微信
E.coli EndonucleaseVIIIfunctionsasbothanN-glycosylase(byexcisingoxidativebaselesions)andanAPlyase(bysubsequentlycleavingthephosphodiesterbackbone),leavingterminalphosphatesatthe5′and3′ends.(1)DamagedbasesremovedbyEndonucleaseVIIIinclude:urea,5,6-dihydroxythymine,thymineglycol,5-hydroxy-5-methylhydanton,uracilglycol,6-hydroxy-5,6-dihydrothymineandmethyltartronylurea(2,3).
SourceofProtein
An E.coli strainwhichcarriestheclonedEndonucleaseVIIIgene.
Suppliedin
10mMTris-HCl
250mMNaCl
0.1mMEDTA
50%glycerol
pH8.0@25°C
SuppliedWith:
B908010XEndonuleaseVlllBuffer
10XEndonucleaseVlllBuffer(B9080)
100mMTris-HCl
750mMNaCl
10mMEDTA
pH8.0@25°C
UnitDefinition
Oneunitisdefinedastheamountofenzymerequiredtocleave1pmolofanoligonucleotideduplexcontainingasingleAPsitein1hourat37°C.
UnitCharacterizationAssay
Specificactivitywasmeasuredusinga2-foldserialdilutionmethod.DilutionsofenzymewerepreparedinEndoVIIIglycerolstoragesolutionandaddedto10µLreactionscontaining2.0µMofanFAM-labeled,34-base,duplexoligonucleotide,containingasingleUracil.[Note:substratepre-treatedfor2minuteswith1unitofUDGtocreateanabasicsite]Reactionswereincubated60minutesat37°C,plungedonice,denaturedwithN-N-dimethylformamideandanalyzedbyrunningandexposingtoshort-waveUVa15%TBE-Ureaacrylamidegel.
SDS-Page(PhysicalPurityAssessment)andSpecifications
2.0µLofconcentratedenzymesolutionwasloadedonadenaturing4-20%Tris-GlycineSDS-PAGEgelflankedbyabroad-rangeMWMarkerand2.0µLofa1:100dilutionofthesample.Followingelectrophoresis,thegelwasstainedandthesamplescomparedtodeterminephysicalpurity.Theacceptancecriteriaforthistestrequiresthattheaggregatemassofcontaminantbandsintheconcentratedsampledonotexceedthemassoftheproteinofinterestbandinthedilutesample,confirminggreaterthan99%purityoftheconcentratedsample.
Single-StrandedExonucleaseActivity
A50µLreactioncontaining15,000cpmofaradiolabeledsingle-strandedDNAsubstrateand10µLofenzymesolutionincubatedfor4hoursat37°Cresultedinlessthan1.0%releaseofTCA-solublecounts.
Double-StrandedExonucleaseActivity
A50µLreactioncontaining15,000cpmofaradiolabeleddouble-strandedDNAsubstrateand10µLofenzymesolutionincubatedfor4hoursat37°Cresultedinlessthan1.0%releaseofTCA-solublecounts.
E.coli 16SrDNAContaminationTest
Replicate5µLsamplesofenzymesolutionweredenaturedandscreenedinaTaqManqPCRassayforthepresenceofcontaminating E.coli genomicDNAusingoligonucleotideprimerscorrespondingtothe16SrRNAlocus.Theacceptancecriterionforthetestisthethresholdcyclecount(Ct)producedbytheaverageof3replicatenotemplatecontrolsamples.BasedonthecorrelationbetweenthenotemplatecontrolCt values,andstandardcurvedata,thedetectionlimitofthisassayis<10copiesgenome/sample.
| EndonucleaseVIII | |
|---|---|
| PartNumber | Y9080L |
| Price | $363 |
| Concentration | 10,000U/ml |
| UnitSize | 10,000U |
| SDS | Availableonrequest |
| StorageTemperature | -25to-15°C |
|---|---|
| Test | Specification |
| Purity(SDS-PAGE) | >99% |
| SpecificActivity | 770,000U/mg |
| SSExonuclease | 10U<1.0%released |
| DSExonuclease | 100U<1.0%released |
| E.coliDNAContamination | 100U<10copies |
*Foradetailedsummaryofassayconditionsanddata,refertotheQualityControlsAnalysissection.
LimitationsofUse
Thisproductwasdeveloped,manufactured,andsoldforinvitrouseonly.Theproductisnotsuitableforadministrationtohumansoranimals.SDSsheetsrelevanttothisproductareavailableuponrequest.